Not applicable.
The present invention is directed to polynucleotides encoding inhibitors of apoptosis. The polynucleotides can be used to control apoptosis in target cells.
Apoptosis denotes a type of programmed cell death in which the cell nucleus shrinks, the genetic material (DNA) progressively degrades, and the cell collapses (see, e.g. Kawabe, et al. Nature 349:245-248 (1991). In many organisms, apoptosis plays an important physiological role in development, tissue homeostasis, eradication of virus-infected cells, and other events (Wyllie, A. H., Kerr, J. F. R. and Currie, A. R. (1980) Int. Rev. Cytol. 68, 251-306)). Caspases are a family of intracellular proteases responsible for execution of the apoptotic program (Salvesen, G. S. and Dixit, V. M. (1997) Cell 91, 443-446). They are initially synthesized as inactive zymogens that are activated by proteolytic processing, generating the requisite large- and small-subunits which comprise the active caspase enzyme. The functional conservation of caspases in inducing apoptosis within various insect, plant, and animal species makes them appropriate targets for influencing the apoptosis process.
Some viruses harbor genes which encode caspase inhibitory proteins, thereby suppressing host defense mechanisms which would otherwise eliminate virus-infected cells by apoptosis. Examples of viral caspase inhibitors include the baculoviral p35 protein (Clem, R. J. et al., (1991) Science 254, 1388-1390 and the crmA protein of the Poxviridae-family cowpox virus (Ray, C., et al., (1992) Cell 69, 597-604). IAP family proteins were first discovered in baculoviruses (Birnbaum, M., et al., (1994) Journal of Virology 68:2521-2525; Crook, N. E., et al., (1993) J. Virol. 67: 2168-2174). Genetic complementation analysis revealed that the Inhibitor of Apoptosis Protein (xe2x80x9cIAPxe2x80x9d) genes of the CpGV and OpMNPV baculoviruses can rescue p35-deficient viruses, maintaining host cell survival so that viral replication successfully occurs (Birnbaum, M., (1994), supra, Crook, N. E., et al., (1993) supra). Baculoviral IAPs contain two tandem copies of a Baculovirus Inhibitory Repeat (BIR) domain followed by a C-terminal RING domain. Mutagenesis studies suggest a requirement for both the BIR and RING domains for their anti-apoptotic function in insect cells. Since these initial discoveries, cellular IAP homologs have been found in many animal species, including Drosophila, mammals, and humans (reviewed in (Miller, L. (1999) Trends in Cell Biology 9:323-328; Deveraux, Q. and Reed, J. C. (1998) Genes Dev. 13:239-252)). All cellular IAPs contain one to three copies of a baculoviral inhibitory repeat (BIR) domain and most also contain a RING domain located near their C-termini. A mechanism for IAP-family proteins was shown when it was reported that several human IAPs, including XIAP, cIAP1, cIAP2, can directly bind and inhibit certain caspases, including caspases-3, -7, and -9 (Deveraux, Q., et al., (1999) EMBO J., in press.; Deveraux, Q. and Reed, J. C. (1998) supra; Deveraux, Q., et al. (1997) Nature 388:300-303; Roy, N., et al., (1997) EMBO J. 16, 6914-6925). Subsequent deletional analysis indicated that the second BIR domain (BIR2) of XIAP is sufficient for inhibiting mammalian caspases-3 and -7 (Roy, N., et al., (1997), supra; Takahashi, R. et al., (1998) J. Biol. Chem. 273, 7787-7790). However, recently it was shown that a fragment of XIAP encompassing the third BIR domain (BIR3) and RING domain specifically inhibits mammalian caspase-9 (Takahashi, R. et al., (1998), supra). Thus, among mammalian IAPs, different regions of these proteins appear to mediate inhibitory interactions with specific caspases.
Though other types of mechanisms have not been excluded, it has been suggested that Drosophila and baculovirus IAPs also may inhibit some caspases. It has been shown that Drosophila IAP1 (DIAP1) is able to inhibit drICE and DCP-1 in insect cells and in yeast (Kaiser, W. et al., (1998) FEBS Lett. 440, 243-248; Hawkins, C., et al., (1999) Proc. Natl. Acad. Sci. USA 96, 2885-2890). It has also been shown that CpIAP and OpIAP require both BIR and RING domains to inhibit activation of Sf-caspase-1 during baculovirus-induced apoptosis in Sf-21 cells. (Seshagiri, S. and Miller, L. K. (1997) Proc. Natl. Acad. Sci. USA 94, 13606-13611.)
Interestingly, a group of apoptosis-inducing genes which encode IAP-binding proteins has been identified in Drosophila, including reaper, hid, and grim (White, E. and Cipriani, R. (1989) Proc. Natl. Acad. Sci. USA 86, 9886-9890). The Reaper, Hid, and Grim proteins contain a homologous 14 amino acid N-terminal domain which is both necessary and sufficient for binding DIAP 1 and for inducing apoptosis (Vucic, D., et al., (1997) Proc. Natl. Acad. Sci. USA 94, 10183-10188; Vucic, D., et al., (1998) Mol. Cell. Biol. 18, 3300-3309). Though initially controversial (reviewed in Deveraux, Q. and Reed, J. C. (1998) Genes Dev. 13, 239-252; Miller, L. (1999) Trends in Cell Biology 9, 323-328), recent data suggest that Reaper, Hid, and Grim induce apoptosis by inhibiting IAPs thus interfering with IAP-mediated suppression of caspases (Wang, S., et al., (1999) Cell 98, 453-463).
The invention provides a cDNA for an Inhibitor of Apoptosis Protein (xe2x80x9cIAPxe2x80x9d) from Spodoptera frugiperda (fall armyworm) (SEQ ID NO:1), as well as nucleic acids that are 85% or more identical to that cDNA. Further, the invention provides polypeptides that are at least 90% identical to a polypeptide encoded by SEQ ID NO:1 (the polypeptide is SEQ ID NO:3). In preferred embodiments, the polypeptides are at least 95% identical to SEQ ID NO:3.
The invention further provides host cells comprising recombinant expression cassettes comprising a promoter operably linked to a polynucleotide at least 85% identical to SEQ ID NO:1, at least 95% identical to SEQ ID NO:1, or that comprises SEQ ID NO:1. The promoter can be inducible or can be constitutive. The host cell can be an insect cell, a plant cell, a mammalian cell. The invention provides recombinant expression cassettes comprising polynucleotides that are 85% or more identical to, 95% or more identical to, or that comprises SEQ ID NO:1. The recombinant expression cassette will typically comprise a promoter, which promoter can be inducible or can be constitutive.
The invention further provides recombinant baculoviruses which have been engineered to contain a nucleic acid which is 85% or more identical to SEQ ID NO:1, which is 95% or more identical to SEQ ID NO:1, or which comprises SEQ ID NO:1. The invention further provides transgenic plants which contain a nucleic acid which is 85% or more identical to SEQ ID NO:1, which is 95% or more identical to SEQ ID NO:1, or which comprises SEQ ID NO:1.
The invention further provides in vitro methods of assaying for compounds capable of specifically binding to an IAP, wherein the method comprises combining an IAP with a test compound and assaying whether the test compound specifically binds to the IAP, where the IAP has a sequence at least 90% or more identical to SEQ ID NO:3. In preferred embodiments, the LAP has a sequence at least 95% identical to SEQ ID NO:3. The IAP can be immobilized on a solid support or can be in an aqueous solution. In some embodiments, the test compound can be bound to a solid support and contacted with the IAP.
The invention further provides in vitro methods of assaying for modulators of IAP activity wherein the method comprises combining an IAP with a test compound and assaying whether the test compound can increase or decrease IAP binding specifically to a capsase polypeptide. The IAP can be immobilized on a solid support or can be in an aqueous solution. In some embodiments, the test compound can be bound to a solid support and contacted with the IAP.
The invention further provides an in vitro method of assaying for the presence of IAP cDNA comprising hybridizing said cDNA to a nucleic acid 85% or more identical to SEQ ID NO:1 or to a contiguous portion of SEQ ID NO:1 at least 20 nucleotides in length and detecting hybridization of the cDNA and the nucleic acid, wherein detection of said hybridization is indicative of the presence of said IAP cDNA. The method further provides an in vitro method of assaying for the presence of an IAP comprising binding the IAP with an antibody which specifically binds said IAP, or with a fragment of said antibody which retains specificity for said IAP, and detecting said binding, wherein said detection is indicative of the presence of the IAP. The detection can be by an ELISA, a Western blot, or other methods known in the art.
Introduction
Apoptosis, or programmed cell death, is an essential process for normal development and homeostasis in multicellular organisms. It plays an important role in disease resistance in organisms as diverse as insects, plants and higher animals, including mammals. In insects, for example, resistance to viral infection includes apoptosis of infected cells, thereby limiting the spread of the invading pathogen. Similarly, in plants, apoptosis of infected cells limits the ability of viral and other infections to spread. This tendency of plants to react to infection by apoptosis of infected cells is in fact exploited by some fungi to create lesions exposing plant tissue more susceptible to fungal invasion. In mammals, such as humans, apoptosis kills cells which have undergone damage to their DNA when they go through the G2/M checkpoint. Many cancers are able to grow because they are able to avoid this control mechanism. Thus, the ability to modulate apoptosis (that is, to increase or to decrease it at will) is useful for modulating the ability of plants and of insects and other animals to pathogens and cancer, Spodoptera frugiperda (fall armyworm) is a lepidopteran host of the Autographa Califomica Nuclear Polyhedrosis Virus (AcMNPV), a member of the baculovirus family. Despite extensive use of S. frugiperda-derived cells for studies of apoptosis-regulation by baculoviruses (reviewed in (Miller, L. (1999) Trends in Cell Biology 9, 323-328)), no endogenous apoptosis-regulating genes have yet been identified in these insect cells, with the exception of Sf-caspases-1 (Ahmad, M., Srinivasula, S., Wang, L., Litwack, G., Fernandes-Alnenri, T. and Alnenri, E. (1997) J. Biol. Chem. 272, 1421-1424).
The present invention demonstrates the cloning and chacterizating of a cellular IAP from S. frugiperda (SfIAP). Sf has been deposited with GenBank and will be publicly available after the filing of this specification under accession number AF186378. SfIAP shares considerable sequence similarity with baculoviral IAPs (vIAPs), suggesting these viruses acquired their vIAP genes from host cells. Analysis of the SfIAP and CpIAP proteins indicates that they are direct inhibitors of mammalian caspase-9, suggesting evolutionary conservation of IAP-family protein functions and providing an explanation for previous reports that baculovirus IAPs can inhibit apoptosis induced by many stimuli in mammalian cells (Hawkins, C. J., Uren, A. G., Hacker, G., Medcalf, R. L. and Vaux, D. L. (1996) Proc. Natl. Acad. Sci. USA 93, 13786-13790; Hawkins, C., Ekert, P., Uren, A., Holmgreen, S. and Vaux, D. (1998) Cell Death and Differentiation 5, 569-576; Uren, A. G., Pakusch, M., Hawkins, C. J., Puls, K. L. and Vaux, D. L. (1996) Proc. Natl. Acad. Sci. USA 93, 4974-4978). Other IAPs are available in GenBank under accession numbers U45880 (human),U75285 (human),U45879 (human), U45878 (human), NM 009689 (mouse), NM 009688 (mouse), L05494 (baculovirus), and L224564 (baculovirus).
Peptides corresponding the N-terminal IAP-binding domain of Grim negate the ability of SfIAP and CpIAP to inhibit mammalian caspases in vitro, providing evidence that mechanisms similar to those described in Drosophila may be used to regulate the IAPs of Spodoptera frugiperda and baculoviruses.
The IAPs of the invention provide a convenient new way to screen for compounds which can modulate apoptosis. Additionally, recombinant viruses encoding IAPs of the invention can be used to infect undesirable insects. The IAPs then decrease the ability of the insects to limit the resulting infection, with increased mortality. Further, IAPs of the invention can be used to transfect plants to render the plants less susceptible to fungi and other organisms which exploit the normal apoptosis response of the plant to render it more vulnerable to invasion. Further, the IAPs of the invention can be used in animals to combat various disorders in which apoptosis plays a role. In preferred embodiments, the animal is a mammal. In particularly preferred uses, the mammal is a human.
The invention can be exploited in a variety of ways. Polypeptides of the invention can be directly administered as therapeutic agents. For use in animals, such as humans, the polypeptides can be administered intravenously, or they can be encapsulated and administered orally. Cells can be transfected with antisense oligonucleotides to bind to IAP-encoding nucleic acids and block their expression. Small chemical compounds which block the binding of IAPs to caspases can be used to interfere with the normal activity of LAPs.
The IAPs of the invention can also be used in vitro to monitor for expression of IAP cDNA. Conveniently, this can be done by immobilizing nucleic acid of an IAP of the invention on a solid substrate, such as a xe2x80x9cchip,xe2x80x9d permitting the nucleic acid to act as a probe to screen a cDNA library or cDNA from a sample of interest. Typically, the length of the nucleic acid from the IAP of the invention used as a probe is chosen to be sufficiently long to permit specific hybridization to a cDNA of a IAP of the invention as opposed to other IAPs. Conveniently, such probes are 20 nucleotides or more, or 25, 30, 35, 40, 45, 50, or even more in length, to permit specific detection of the target cDNAs.
In another set of embodiments, the IAPs of the invention can be used to detect and monitor the presence of IAPs in a sample. Typically, antibodies are generated against an IAP of the invention. Depending on the purpose of the assays, the antibodies can be such that they detect all IAPs, or the antibodies can be absorbed against other known IAPs to eliminate antibodies which recognize IAPs other than those of the invention. Methods of generating antibodies against antigens are well known in the art. Typically, monoclonal antibodies are preferred. Fragments of antibodies which retain binding specificity for the IAP can also be used. Fragments such as single chain Fvs (scFvs), disufide stabilized Fvs (dsFvs), Fabs, and Fab""s are well known in the art. Assays employing such antibodies, such as ELISAs and Western blots, are also well known in the art.
Definitions
xe2x80x9cIAPxe2x80x9d refers generally to an inhibitor of apoptosis protein and, more specifically herein, to an inhibitor of apoptosis protein of the invention. Which use is intended will be clear in context. IAPs of the invention typically are at least substantially identical to SEQ ID NO:3. xe2x80x9cSfIAPxe2x80x9d specifically denotes an IAP of the invention cloned from Spodoptera frugiperda (fall armyworm). All cellular IAPs contain one to three copies of a baculoviral inhibitory repeat (BIR) domain and most also contain a RING domain located near their C-termini. SfIAP contains two BIR domains, followed by a RING domain near its C-terminus. Within the BIR and RING regions, SfIAP shares 85% amino acid identity (90% similarity) with baculoviral CpIAP and 70% identity (80% similarity) with OpIAP.
xe2x80x9cApoptosisxe2x80x9d refers to programmed cell death. It is an essential process for normal development and homeostasis in multicellular organisms such as mammals, insects and plants. Apoptosis differs from general cell death as from a toxin or ischemic event in that the cell is shutting down according to a controlled pattern of events.
xe2x80x9cApoptosis-inducing chemicalxe2x80x9d refers to a chemical that promotes programmed cell death. The chemical can be a administered directly to the plant or indirectly by secretion of a pathogen.
xe2x80x9cHeterologous genexe2x80x9d refers to a gene introduced into a host cell via recombinant technology where that gene is either not naturally present in the cell or represents an additional copy of an endogenous gene or is operably linked to a promoter that is not normally found in association with the gene in the host cell.
xe2x80x9cInducible plant promoterxe2x80x9d refers to a promoter which directs expression of a gene where the level of expression is alterable by factors such as temperature, pH, transcription factors and chemicals.
xe2x80x9cInsecticidal proteinxe2x80x9d refers to a diverse group of compounds that are lethal to insects when ingested. Examples include the crystal proteins or delta endotoxins from Bacillus thuringiensis. 
xe2x80x9cViral plant disease resistancexe2x80x9d refers to the ability of a plant to prevent or inhibit a viral pathogen from successfully infecting it.
xe2x80x9cOperably linkedxe2x80x9d refers to nucleotide sequences which are joined in such a manner that their individual function complements each other. Examples are promoters, transcription terminators, enhancers or activators and heterologous genes which when transcribed and if appropriate to translate will produce a functional product, i.e. a protein, ribozyme or anti-sense construct.
xe2x80x9cPlantxe2x80x9d includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
xe2x80x9cPlant pathogensxe2x80x9d refer to fungi, bacteria, virus and insects that infect plants and have negative effects on their growth and health.
xe2x80x9cPromoterxe2x80x9d refers to a region of DNA involved in binding the RNA polymerase to initiate transcription.
xe2x80x9cTransformation ratexe2x80x9d refers to the percent of cells that are successfully incorporate a heterologous gene into its genome and survive.
xe2x80x9cTransfectingxe2x80x9d refers to the process of introducing a heterologous gene into a cell.
The term xe2x80x9cproteinxe2x80x9d is used herein interchangeably with xe2x80x9cpolypeptidexe2x80x9d and xe2x80x9cpeptide.xe2x80x9d
xe2x80x9cNucleic acidxe2x80x9d refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, hosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl hosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
A polynucleotide sequence comprising a fusion protein of the invention hybridizes under stringent conditions to each of the nucleotide sequences encoding each individual polypeptide of the fusion protein. The polynucleotide sequences encoding the individual polypeptides of the fusion polypeptide therefore include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs.
xe2x80x9cPercentage of sequence identityxe2x80x9d is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term xe2x80x9csubstantial identityxe2x80x9d of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 85% sequence identity. Alternatively, percent identity can be any integer from 85% to 100%. More preferred embodiments include at least: 85%, 90%, 95%, or 99% or higher, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. xe2x80x9cSubstantial identityxe2x80x9d of amino acid sequences for these purposes normally means sequence identity of at least 87%. Preferred percent identity of polypeptides can be any integer from 87% to 100%. More preferred embodiments include at least 87%, 90%, 95%, or 99% identity. Polypeptides which are xe2x80x9csubstantially similarxe2x80x9d share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math. 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.
A preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or to a third nucleic acid, under moderately, and preferably highly, stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993) (Elsevier Science, Inc., New York). Generally, stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.
Exemplary stringent hybridization conditions can be as following: 50% formamide, 5xc3x97 SSC, and 1% SDS, incubating at 42xc2x0 C., or, 5xc3x97 SSC, 1% SDS, incubating at 65xc2x0 C., with wash in 0.2xc3x97 SSC, and 0.1% SDS at 65xc2x0 C.
For the purpose of the invention, suitable xe2x80x9cmoderately stringent conditionsxe2x80x9d include, for example, prewashing in a solution of 5xc3x97SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridizing at 50xc2x0 C.-65xc2x0 C., 5xc3x97 SSC overnight, followed by washing twice at 65xc2x0 C. for 20 minutes with each of 233 , 0.5xc3x97 and 0.2xc3x97 SSC (containing 0.1% SDS). Such hybridizing DNA sequences are also within the scope of this invention.
Screening for IAP Binding Activity
The invention provides in vitro and in vivo methods of assaying for a modulator of IAP activity by identifying molecules that specifically bind an IAP, thereby affecting its activity. While the invention is not limited by what means the IAP activity is inhibited, specific embodiments include assaying for IAP binding to members of the caspase family, as described herein. The methods of the invention also include screening for antibodies directed to an IAP or small molecule binders of IAPs. To assay for specific binding of a putative modulatory molecule, the IAP can be in solution or can be attached to a fixed substrate. In some embodiments, an IAP is fixed to a solid substrate for high throughput screenings or column chromatography.
High-Throughput Screening of Candidate Agents that Bind IAPs of the invention
Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a xe2x80x9clead compoundxe2x80x9d) with some desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. The current trend is to shorten the time scale for all aspects of drug discovery. Because of the ability to test large numbers quickly and efficiently, high throughput screening (HTS) methods are replacing conventional lead compound identification methods.
In one embodiment, high throughput screening methods are used to identify compositions that specifically bind an IAP and modulate its activity. This involves providing a library containing a large number of potential compounds (candidate compounds). Such xe2x80x9ccombinatorial chemical librariesxe2x80x9d are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that bind IAP. The compounds thus identified can serve as conventional xe2x80x9clead compoundsxe2x80x9d or can themselves be used as potential or actual reagents.
Combinatorial chemical libraries
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical xe2x80x9cbuilding blocksxe2x80x9d such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks can result in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (see, e.g., Gallop (1994) 37:1233-1250).
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton (1991) Nature, 354: 84-88). Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention. Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, Oct. 14, 1993), random bio-oligomers (PCT Publication WO 92/00091, Jan. 9, 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann (1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic syntheses of small compound libraries (Chen (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho (1993) Science 261:1303), and/or peptidyl phosphonates (Campbell (1994) J. Org. Chem. 59: 658). See, generally, Gordon (I994) J. Med. Chem. 37:1385, nucleic acid libraries, peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see, e.g., Vaughn (1996) Nature Biotechnol 14:309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang (1996) Science 274:1520-1522, and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) CandEN, Jan 18, page 33, isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, orpholino compounds U.S. Pat. Nos. 5,506,337, benzodiazepines 5,288,514).
Devices for the preparation of combinatorial libraries are commercially available; see, e.g., 357 MPS, 390 NPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.; the Ultra-high Throughput Screening System (UHTSS(trademark)) capable of screening over 100,000 compounds per day, Aurora BioSciences, San Diego, Calif.
A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.).
High throughput assays of chemical libraries
Any of the assays for compounds capable of binding IAPs and/or modulating IAP activity described herein are amenable to high throughput screening. These systems (examples of which are described above) can automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization.
Attaching of the IAP to a Solid Support
IAP, whether full length, or subsequences thereof (e.g., a BIR domain or RING domain) can be bound to a variety of solid supports. Solid supports that can be used in the methods of the invention include polymer beads, membranes (e.g., nitrocellulose or nylon), microtiter dishes (e.g., PVC or polystyrene), test tubes, dip sticks (e.g., glass, PVC, polypropylene, and the like), microfuge tubes, glass, silica, plastic, metallic beads, or substrates such as paper.
Adhesion of a IAP xe2x80x9ctargetxe2x80x9d molecule to the solid support can be direct (i.e. directly contacting the solid support) or indirect (a particular compound or compounds are bound to the support and IAP binds to this compound rather than the solid support). Immobilization of compounds can be covalent, e.g., utilizing single reactive thiol groups of cysteine residues (see, e.g., Colliuod (1993) Bioconjugate Chem. 4:528-536). Alternatively, compounds can be immobilized non-covalently but specifically, e.g., via immobilized antibodies (see above), as described by Schuhmann (1991) Adv. Mater. 3:388-391; Lu (1995) Anal. Chem. 67:83-87; or, the biotin/strepavidin system, see, e.g., Iwane (1997) Biophys. Biochem. Res. Comm. 230:76-80); or metal chelating, e.g., Langmuir-Blodgett films (Ng (1995) Langmuir 11:4048-4055; Schmitt (1996) Angew. Chem. Int. Ed. Engl. 35:317-20; Frey (1996) Proc. Natl. Acad. Sci. USA 93:4937-41; Kubalek (1994) J. Struct. Biol. 113:117-123; or, metal-chelating self-assembled monolayers, see, e.g., Sigal (1996) Anal. Chem. 68:490-497, for binding of polyhistidine fusion proteins.
Indirect binding of IAP can be achieved using a variety of linkers, many of which are commercially available. The reactive ends can be any of a variety of functionalities, e.g., amino reacting ends such as N-hydroxysuccinimide (NHS) active esters, aldehydes, epoxides, isocyanate, and nitroaryl halides; and thiol reacting ends such as pyridyl disulfides, maleimides, and active halogens. The heterobifunctional crosslinking reagents have two different reactive ends, e.g., an amino-reactive end and a thiol-reactive end, while homobifunctional reagents have two similar reactive ends, e.g., bismaleimidohexane (BMH) which permits the cross-linking of sulfhydryl-containing compounds. The spacer can be aliphatic or aromatic. Examples of commercially available homobifunctional cross-linking reagents include, but are not limited to, the imidoesters such as dimethyl adipimidate dihydrochloride (DMA), dimethyl pimelimidate dihydrochloride (DMP); and dimethyl suberimidate dihydrochloride (DMS). Heterobifunctional reagents include commercially available active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) and the sulfosuccinimidyl derivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce Chemicals, Rockford, Ill.). Another group of coupling agents is the heterobifunctional and thiol cleavable agents such as N-succinimidyl 3-(2-pyridyidithio)propionate (SPDP) (Pierce).
By manipulating the solid support and the mode of attachment of the target IAP molecule to the support, it is possible to control the orientation of the IAP. Thus, for example, it is possible to attach the IAP molecule to a surface in a manner that leaves a xe2x80x9ctailxe2x80x9d free to interact with other molecules, e.g., a IAP fusion protein with a non-IAP tag e.g., FLAG, myc, GST, polyHis, etc.) for attachment to the column.
Once bound there are a variety of assay formats that can be used to screen for modulators of the IAP. For example, molecules that interact with a IAP binding domain can be identified by attaching the IAP to a solid support, contacting a second molecule with the support coated with IAP, and detecting the binding of the second molecule to the IAP. Molecules that interact or bind with the target are then eluted, thereby isolating molecules that interacted with the IAP.
Assays
A variety of different assays for detecting compounds and compositions capable of binding IAP can be used in this invention. For a general description of different formats for binding assays, see BASIC AND CLINICAL IMMUNOLOGY, 7th Ed. (D. Stiles and A. Terr, ed.)(1991); ENZYME IMMUNOASSAY, E. T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980); and xe2x80x9cPractice and Theory of Enzyme Immunoassaysxe2x80x9d in P. Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, Elsevier Science Publishers, B.V. Amsterdam (1985).
In competitive binding assays, the test compound competes with a second compound (known to specifically bind IAP) for specific binding sites on the IAP molecule attached to the solid support. Binding is determined by assessing the amount of second compound associated with the fixed IAP molecule. The amount of second compound associated with IAP is inversely proportional to the ability of a test compound to compete in the binding assay.
The amount of inhibition or stimulation of binding of a labeled second compound by the test compound depends on the binding assay conditions and on the concentrations of labeled analyte and test compounds used. Under specified assay conditions, a test compound is said to be capable of inhibiting the binding of a second compound to a IAP target compound if the amount of bound second compound is decreased by 50% or more compared to a control (no test compound) sample.
Alternatively, various known or unknown compounds, including proteins, carbohydrates, and the like, can be assayed for their ability to directly, and specifically, bind to the target immobilized IAP. In one embodiment, samples from various tissues are contacted with IAP. In another embodiment, small molecule libraries and high throughput screening methods are used to identify compounds that bind to the target. The IAP-binding molecules is then eluted using any method, e.g., column chromatography techniques.
Isolation of nucleic acids of the invention
The nucleic acids of the invention can be used to prepare recombinant proteins and transgenic organisms for a number of purposes. Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer""s specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley and Sons, Inc. (1994-1998).
The isolation of nucleic acids may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
The genomic library can then be screened using a probe based upon the sequence of a cloned IAP gene disclosed here. Probes may be used to hybridize with genomic DNA sequences to isolate homologous genes in the same or different species. Alternatively, antibodies raised against a polypeptide such as an IAD of the invention can be used to screen an expression library. Making antibodies against an antigen of choice is well known in the art.
Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of IAP genes directly from genomic DNA or from cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired RNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). Appropriate primers and probes for identifying sequences from tissues are generated from comparisons of the sequences provided here (e.g. SEQ ID NO: 1, etc.).
Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Expression in prokaryotes and eukaryotes
To obtain high level expression of a cloned gene, such as apolynucleotides encoding IAPs of the invention, one typically subclones polynucleotides encoding the IAP into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systems for expressing the IAP are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
In addition to the promoter, the expression vector typically contains transcription unit or expression cassette that contains all the additional elements required for the expression of the IAP encoding nucleic acid in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding IAP and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
In addition to apromoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc.
Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a IAP encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of the IAP, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss and Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing IAP.
After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of IAP, which is recovered from the culture using standard techniques. A number of procedures can be employed when recombinant polypeptides are being purified. For example, proteins having established molecular adhesion properties can be reversible fused to the polypeptides. With the appropriate ligand, the polypeptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally the polypeptides could be purified using immunoaffinity columns.
Production of transgenic plants
The IAPs of the invention can be used for increasing plant disease resistance. More specifically, IAPs can be used to delay, suppress or inhibit an apoptosis response in plants. Many plant pathogens, in particular, non-viral plant pathogens, induce apoptosis in plants as a part of the infection process. When a plant is transformed with IAP of the invention, the plant will not be as susceptible to pathogen induced apoptosis and a resistance phenotype is generated.
DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. (1984) EMBO J. 3:2717-2722. Electroporation techniques are described in Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824. Ballistic transformation techniques are described in Klein et al. (1987) Nature 327:70-73.
Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995).
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased seed mass. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
The nucleic acids of the invention can be used to confer desired traits on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.
One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Using known procedures one of skill can screen for plants of the invention by detecting the increase or decrease of IAP mRNA or protein in transgenic plants. Means for detecting and quantitating mRNAs or proteins are well known in the art.
In addition, modulation of apoptosis in plant cells can be monitored. Apoptosis in plants follows an analogous pathway as in animal cells. Two molecular analytic tools are available and established to determine when apoptosis is occurring in plants (as well as animal cells) as distinguished from cell death due to lethal toxins (such as toxic levels of Fe2SO4) or hypoxic conditions. The first is demonstration of an orderly fragmentation of the cellular DNA. These are called DNA ladders and are readily analyzed using a variety of electrophoretic techniques. The specific gel or running conditions are not critical. Suitable electrophoretic conditions are provided by Wang et al. (1996), Cell, 8:375-391.
Alternatively, fragmentation of DNA during apoptosis can be detected in situ by reagents that react with exposed 3xe2x80x2hydroxyl groups on the nucleosomal units. The assay procedure involves end labeling the DNA fragments by terminal deoxynucleotidyl transferase (TdT) with UTP conjugated to a detectable marker. The method is termed (TUNEL) and more details of this method can be found in Wang et al. (supra). Finally one can visually identify apoptotic bodies containing fragmented DNA which is one of the hallmarks of apoptosis.
One of skill can also assay for disease resistance by traditional methods. These methods involve visual observation of the effects of a pathogen on a host plant. Observable symptoms include wilt, rot, stunted growth, color changes and mycelial growth. The symptoms are scored for their degree of intensity in sufficient repeated trials until statistically meaningful information is generated. The generic experimental parameters for reproducible study of infection and resistance in plants is well known.
Preparation of recombinant plant vectors
To use isolated sequences to transform plants, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the desired polypeptide, for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.
For example, for overexpression, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as xe2x80x9cconstitutivexe2x80x9d promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1xe2x80x2- or 2xe2x80x2-promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill. Such genes include for example, ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. XI 5596, Martinez et al. J. Mol. Biol 208:551-565 (1989)), and Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)).
Alternatively, the plant promoter may direct expression of nucleic acid in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control (i.e. inducible promoters). Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, the presence of light, or sprayed with chemicals/hormones. In a preferred embodiment, the promoter is specifically induced by infection of a pathogen. Tissue-specific promoters can be inducible. Similarly, tissue-specific promoters may only promote transcription within a certain time frame of developmental stage within that tissue. Other tissue specific promoters may be active throughout the life cycle of a particular tissue. One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.
A number of tissue-specific promoters can also be used in the invention. For instance, promoters that direct expression of nucleic acids in leaves, roots or flowers are useful for enhancing resistance to pathogens that infect those organs. For expression of a IAP in the aerial vegetative organs of a plant, photosynthetic organ-specific promoters, such as the RBCS promoter Khoudi, et al., Gene 197:343, 1997), can be used. Root-specific expression of IAP polynucleotides can be achieved under the control of the root-specific ANR1 promoter (Zhang and Forde, Science, 279:407, 1998). Any strong, constitutive promoters, such as the CaMV 35S promoter, can be used for the expression of IAP polynucleotides throughout the plant.
If proper polypeptide expression is desired, a polyadenylation region at the 3xe2x80x2-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
The vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.
Expression in recombinant Baculovirus
Another use of the IAP polynucleotides is in the construction of recombinant baculoviruses that are useful as insecticides. It is known that apoptosis is one of the mechanisms by which insects defend against baculoviral infection. Thus, recombinant baculoviruses comprising the IAP polynucleotides of the invention can be useful in overcoming this defense mechanism.
The construction of most baculovirus expression vector systems has been based on replacement of the polyhedrin gene or other coding region with a foreign gene under the transcriptional control of the polyhedrin gene promoter or other promoter (Pennock, et al., Mol. and Cell. Bio. 4:399-406 (1984); and Smith, et al. Molecular and Cell. Biol. 3:2156-2165(1983)). Baculoviruses are natural pathogens of many agriculturally important insect pests (Wood, and Granados, Ann. Rev. Microbiol. 45:69-87 (1991)). There has thus been an increased interest in exploiting recombinant baculoviruses to express foreign proteins which would improve the pesticidal properties of the native baculoviruse (see, e.g. Carbonell et al., Gene 73:409-418 (1988); Hammock, et al., Nature 344:458-461 (1990); Maeda, S., Biochem. Biophys. Res. Comm. 165: 1177-1183 (1989); Maeda, et al., Virol. 184:777-780 (1991) and U.S. Pat. No. 5,908,785).
Administration of IAP Inhibitors, IAPs, and IAP Nucleic Acids and Anti-IAP Nucleic Acids As Pharmaceuticals
Apoptosis plays a major role in development, viral pathogenesis, cancer, autoimmune diseases and neurodegenerative disorders. Inappropriate increases in apoptosis may cause or contribute to a variety of diseases, including AIDS, neurodegenerative diseases (e.g. Alzheimer""s Disease, Parkinson""s Disease, Amyotrophic Lateral Sclerosis (ALS), retinitis pigmentosa and other diseases of the retina, myelodysplastic syndrome (e.g., aplastic anemia), toxin-induced liver disease (e.g., alcoholism) and ischemic injury (e.g., myocardial infarction, stroke, and reperfusion injury). In addition, disruption of normally occurring apoptosis has been implicated in the development of some cancers (e.g. follicular lymphoma, p53 carcinomas, and hormone dependent tumors), autoimmune disorders (e.g., lupus erythematosis and multiple sclerosis) and viral infections (e.g., herpes virus, poxvirus, and adenovirus infections). See, e.g., Korneluk, U.S. Pat. No. 5,919,912.
The invention provides modulators (e.g., inhibitors) of IAP activity and their therapeutic administration. These compounds include those found by the methods of the invention. Modulators that can be used therapeutically also include antibodies and small molecules which bind to IAP to inhibit its ability to bind caspaces. In another embodiment, the modulator is a peptide inhibitor of IAP activity. The peptides, polypeptides and other compositions of the invention are administered with a pharmaceutically acceptable carrier(s) (excipient) to form the pharmacological composition.
Pharmaceutically acceptable carriers and formulations, e.g., for peptides and polypeptides, are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington""s Pharmaceutical Science, Mack Publishing Company, Easton, Pa. (xe2x80x9cRemington""sxe2x80x9d); Banga; Putney (1998) Nat. Biotechnol. 16:153-157; Patton (1998) Biotechniques 16:141-143; Edwards (1997) Science 276: 1868-1871; Ho, U.S. Pat. No. 5,780,431; Webb, U.S. Pat. No. 5,770,700; Goulmy, U.S. Pat. No. 5,770,201.
The compositions used in the methods of the invention can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or locally; by intraarterial, intrathecal (IT), intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa). Actual methods for delivering compositions will be known or apparent to those skilled in the art and are described in detail in the scientific and patent literature, see e.g., Remington""s.
The pharmaceutical compositions can be administered by any protocol and in a variety of unit dosage forms depending upon the method of administration, whether it is being co-administered a chemotherapeutic agent, and the like. Dosages for typical peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisorial in nature and are adjusted depending on a variety of factors, such as the particular therapeutic context, patient health and the like. The amount of composition or peptide adequate to generate the desired response is defined as a xe2x80x9ctherapeutically effective dose.xe2x80x9d The dosage schedule and amounts effective for this use, i.e., the xe2x80x9cdosing regimen,xe2x80x9d will depend upon a variety of factors, including the stage of the disease being treated; timing of co-administration of other agents; the general state of the patient""s health; the patient""s physical status; age; the pharmaceutical formulation, and the like. The dosage regimen also takes into consideration pharnacokinetics, e.g., the peptide pharmaceutical composition""s rate of absorption, bioavailability, metabolism, clearance, and the like, see, e.g., Remington.
Dosages can be determined empirically, e.g, by abatement or amelioration of symptoms, or by objective criteria, analysis of blood or histopathology specimens (amount of apoptosis in a biopsy), and the like.
Vectors used for therapeutic administration of IAP-encoding nucleic acids or anti-IAP nucleic acids, such as antisense molecules, may be viral or nonviral. Viral vectors are usually introduced into a patient as components of a virus. Illustrative viral vectors into which one can incorporate nucleic acids include, for example, adenovirus-based vectors (Cantwell (1996) Blood 88:4676-4683; Ohashi (1997) Proc. Nat""l. Acad. Sci USA 94:1287-1292), Epstein-Barr virus-based vectors (Mazda (1997) J. Immunol. Methods 204:143-151), adenovirus-associated virus vectors, Sindbis virus vectors (Strong (1997) Gene Ther. 4: 624-627), herpes simplex virus vectors (Kennedy (1997) Brain 120: 1245-1259) and retroviral vectors (Schubert (1997) Curr. Eye Res. 16:656-662).
Nonviral vectors encoding products useful in gene therapy can be introduced into an animal by means such as lipofection, biolistics, virosomes, liposomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA injection, artificial virions, agent-enhanced uptake of DNA, ex vivo transduction. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam(trademark) and Lipofectin(trademark)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424 and WO 91/16024. Naked DNA genetic vaccines are described in, for example, U.S. Pat. No. 5,589,486.